Streptococcus sanguinis is a gram-positive, non-motile bacterium native to human mouths. It is the primary cause of endocarditis and is also responsible for tooth decay. Two-component systems (TCSs) are commonly found in bacteria. In response to environmental signals, TCSs may regulate the expression of virulence factor genes. Gene co-expression networks are exploratory tools used to analyze system-level gene functionality. A gene co-expression network consists of gene expression profiles represented as nodes and gene connections, which occur if two genes are significantly co-expressed. An adjacency function transforms the similarity matrix containing co-expression similarities into the adjacency matrix containing connection strengths. Gene modules were determined from the connection strengths, and various network connectivity measures were calculated. S. sanguinis gene expression profile data was loaded for 2272 genes and 14 samples with 3 replicates each. The soft thresholding power β=6 was chosen to maximize R2 while maintaining a high mean number of connections. Nine modules were found. Possible meta-modules were found to be: Module 1: Blue & Green, Module 2: Pink, Module 3: Yellow, Brown & Red, Module 4: Black, Module 5: Magenta & Turquoise. The absolute value of module membership was found to be highly positively correlated with intramodular connectivity. Each of the nine modules were examined. Two methods (intramodular connectivity and TOM-based connectivity followed by network mapping) for identifying candidate hub genes were performed. Most modules provided similar results between the two methods. Similar rankings between the two methods can be considered equivalent and both can be used to detect candidate hub genes. Gene ontology information was unavailable to help select a module of interest. This network analysis would help researchers create new research hypotheses and design experiments for validation of candidate hub genes in biologically important modules.

L'activitat de grup sanguini A en els eritròcits, quan aquesta ha estat referida a substàncies no lipídiques, s'ha assignat de forma concreta a la glicoforina A, la glicoproteïna més gran present en la membrana, i també a l'anomenada banda 3 la principal proteïna de l'eritròcit. En altres casos l'activitat de grup sanguini ha estat relacionada amb altres glicoproteïnes sense especificar-ne els trets més característics.En base a aixó, l'objectiu de la tesi ha estat la caracterització fisico-química i estructura d'una molècula que químicament tingui naturalesa glicoproteïnica, anant acompanyada aquesta d'activitat de grup sanguini A.Així, a partir de sang de 18 donants sans s'han obtingut les membranes de l'eritròcit per mitjà de la lisi hipotònica. La presència d'activitat acetilcolinesterasa en les membranes indica que les vesícules s'han tancat amb la mateixa orientació que tenien els eritròcits abans de la lisi. Les membranes, un cop homogenitzades, dialitzades i liofilitzades, s'han sotmes al procés de solublització, és a dir, el procés que converteix l'estat complexe de la membrana intacta en un estat més senzill que permeti l'anàlisi dels components de la membrana per mètodes que no poden aplicar-se a la membrana intacta.Després de realitzar un estudi comparatiu amb tres tècniques solubilitzadores, s'ha escollit la fonamentada amb l'extracció dels lípids per la mescla cloroform-metanoI (2:1, v/v). A continuació, les glicoproteïnes es precipiten de la fase aquosa per adició d'etanol (concentración final del 60 %) i manteniment durant 48 h a -20°C en presència de clorur sòdic.Les glicoproteïnes es purifiquen per cromatografia de filtració per gel (Sephadex G-75) i per cromatografia d'afinitat fent passar les glicoproteïnes a través d'un gel que contenia la lectina Helix pomatia (Helix pomatia Lectin - Sepharose 6MB), que específicament uneix les glicoproteïnes que porten com a sucre terminal la N-acetil-D-galactosamina, glúcid característic de les glicoproteïnes amb activitat de grup sanguini A.La glicoproteïna eluïda de la columna per una concentración 0.1 M de N-acetil-D-galactosamina té una capacitat inhibitòria de l'hemaglutinació de 38.82 micrograms/mL, amb una forquilla de 39.43 i 38.21 micrograms/mL, per un Iímlt de confiança alfa=90. L'anàlisi dels aminoàcids i glúcids corresponents ha proporcionat uns percentatges respectius de proteïna i sucres del 41.05 i 58.95. Cal destacar l'alta presència de serina i treonina (aminoàcids que en una glicoproteïna són els que uneixen la cadena glucídica al nucli peptídic), la molt baixa proporció d'aminoàcids cromòfors (triptofan i tirosina), la qual cosa explica que la molècula no presenti una marcada absorció pels voltants dels 280 nm i també justifica el baix valor del coeficiente d'absorció específica (0.4324 L/g. cm). La molècula no conté cisteïna i, per tant, no poden existir enllaços disulfuro intramoleculars. Pel que fa als glúcids, la meitat d'aquestos corresponen a hexosamines, trobant-s'hi també hexoses neutres i àcid siàlic.La valoració de la glicoproteïna pel mètode clàssic de Lowry presenta una gran pèrdua de sensibilitat envers la que el mètode presenta quan hom valora una proteïna estàndard, com és ara el cas de I'albúmina sèrica bovina.Per aixó, s'ha desenvolupat un mètode per a quantificar les glicoproteïnes basat en determinar per espargiment de la llum ("light scattering") la turbidesa produïda per l'adició d'etanol a la solució de glicoproteïna. Aquest mètode per l'afició d'etanol a la solució de glicoproteïna. Aquest mètode, a més de precís, no és destructiu.El valor de la masa molecular calculat per a la glicoproteïna ha estat de 53500 ±4280 Da (per light scattering), de 41500 Da (per anàlisi d'aminoàcids) i de 76000 la forma dimèrica i 43000 la monomèrica (per PAGE-SDS). La cromatografia de filtració per gel no és adient per a determinar la masa molecular atès que proporciona valors molt superiors als reals a causa de l'efecte distorsionant de l'àcid siàlic i de la possible capacitat d'autoagregació.Entre les propietats físico-químiques estudiades (absorció a l'espectre U.V., dependència de la turbidesa amb la longitud d'ona, espectroscòpia derivativa, variación de l'espectre d'absorció a l'U.V. en funció del pH, ionització dels grups fenòlics, absorció a l'espectre I.R., dispersió rotatòria òptica (D.R.O.), comportament cromatogràfic, viscositat, punts isoelèctric i isoiònic, volum específic parcial, increment de l'índex de refracció i solubilitat), destaquen aquelles que proporcionen una més gran informació sobre la molècula, cas de la D.R.O. i de la viscositat. L'espectre de D.R.O. de la glicoproteïna presenta una vall negativa de feble magnitud a 234 nm, mentre que els valors de la viscositat intrínseca i de la constant d'Huggins apunten vers una estructura de la molècula en cabdell monoestadístic ("random coil").A partir de les característiques estructurals determinades s'ha pogut obtenir que la glicoproteïna té una estructura molt expandida (37.70 nm de radi de gir), que presenta un 50 % de cabdell monoestadístic, un 30 % d'alfa-helix i un 20 % de beta-fulla (segons els valors obtinguts entre 220 i 250 nm, aplicant els tractaments de Greenfield et al. i Chen et al.), i que els aminoàcids cromòfors es troben tant en l'interior com en l'exterior de la molècula (estudi de l'acció dels agents pertorbants no polars: urea, d,ioxà i polietilenglicol). Finalment, s'ha estudiat per diferents tècniques l'estabilitat estructural de la glicoproteïna enfront del clorhidrato de guanidina, la urea i la calor. Amb els resultats obtinguts pot arribar-se a la conclussió que la glicoproteïna és molt estable; que, a més, l'acció desnaturalitzant és reversible i que el procés de desnaturalització és complexe, no podent ésser identificat amb un procés en dos estats, natiu i desnaturalitzant, com succeeix en moltes proteïnes. / "DETERMINATION OF ESTRUCTURAL PARAMETERS FROM A GLYCOPROTEIN BEARING BLOOD GROUP A ACTIVITY"TEXT: A physicochemical, structural study about a human blood group-A glycoprotein (GP) has been carried out. GP was purified through an affinity column, which contains the lectin Helix pomatia. The main chemical characteristics are: low content of chromophores aminoacids, Jack of cysteine, high content of serine and threonine, and of hexosamines, too.Owing to the chemical properties of the GP, the Lowry's method valoration is not suitable, and for this reason, it has been developed a new method based on quantifying by light scattering the turbidity that is produced by the addition of absolute ethanol.The molecular mass of GP is 53500 (determined by light scattering), or 41500 (amino acid analysis), while it presents a 76000 Da dimer form and a 43000 Da monomer form by PAGE-SDS.The following physicochemical parameters have been determined: absorption in the U.V. spectrum, derivative spectroscopy, variation of spectrum as pH function, ionization of phenolic groups, absorption in the I.R. spectrum, optical rotatory dispersion, viscosity, isoionic and isoelectric points, partial specific volume, solubility and increase of index refraction. Beyond that, the gyration radius, conformation and the action of disturbing agents (urea, dioxane and polyethyleneglycol) have been studied.Eventually, the structural stability of GP has been determined in front of guanidine hydrochloride, urea and heat. The results show the GP has a mainly random coil structure, and in this fact, there is a great concordance among the values obtained with O.R.D., viscosity... On the other hand, the molecule is very stable and the denaturalization process is reversible and not involves a mechanism in two steps.

La enfermedad cardiovascular es un trastorno complejo, multifactorial, poligénico y con un importante componente ambiental. Este grupo de enfermedades, y en particular la cardiopatía isquémica, ocasionan una gran morbilidad y mortalidad a nivel poblacional. Por lo tanto, la prevención es un elemento importante en todas las políticas de salud pública. En la última década se ha avanzado de manera notable en el conocimiento del componente genético de la cardiopatía isquémica y se han descubierto más de 50 loci asociados con el riesgo de presentar esta enfermedad. Muchos de estos loci no se asocian con los factores de riesgo cardiovascular clásicos, y el mecanismo etiopatogénico que explica esta asociación no se conoce. Por otra parte, la variabilidad genética tambén puede explicar parte de la variabilidad interindividual en la respuesta a diferentes fármacos y asociarse al pronóstico de la enfermedad. / Cardiovascular disease is a complex, multifactorial, polygenic disorder with an important environment component. This group of diseases, especially ischemic heart disease, causes significant morbidity and mortality at the population level. Therefore, prevention is a key element of relevant public health policies. Over the past decade there has been a significant advance in knowledge of the genetic component of ischemic heart disease; more than 50 loci associated with risk for this disease have been discovered. Many of these loci are not associated with traditional cardiovascular risk factors, and the etiopathogenic mechanism that explains this association is unknown. Moreover, genetic variability may explain part of the interindividual variability in response to different drugs and may be associated with prognosis.

Cardiovascular disease and, specifically, atherothrombosis is a global health problem with huge devastating consequences. While cardiovascular research has progressed rapidly over the last years, there is still a need for clinically applicable tools for risk prediction, diagnosis, or therapeutic interventions; not only in order to improve earlier identification of cardiac diseases and the prediction of specific therapies avoiding invasive diagnostic procedures and unnecessary treatments, but also to further amplify the understanding of basic mechanisms responsible for their pathogenesis. This thesis mainly focuses on the role of cell-derived microparticles in atherothrombosis, showing their direct effect in the context of arterial thrombosis and investigating their association to preclinical atherosclerosis as a form to exploit them as potential biological markers of disease. The combination of functional, molecular, proteomic and genomic approaches allowed the elucidation of different important aspects of the microparticles both as an interesting therapeutic target and as a novel promising biomarker of silent atherothrombotic disease. / La enfermedad cardiovascular y, específicamente, la aterotrombosis es un problema de salud global con enormes consecuencias devastadoras. Aunque la investigación cardiovascular ha progresado rápidamente durante los últimos años, aún se requieren herramientas aplicables a la clínica para la predicción del riesgo, el diagnóstico o la intervención terapéutica, con el objetivo no solo de mejorar la identificación precoz de las enfermedades cardíacas y la elección de terapias específicas, evitando procedimientos diagnósticos invasivos y tratamientos innecesarios, sino también para ampliar el conocimiento de los mecanismos básicos responsables de su patogenia. La presente tesis se centra principalmente en el papel de las micropartículas celulares en la aterotrombosis, poniendo en evidencia su participación directa en el contexto de la trombosis arterial y asociación con la aterosclerosis preclínica, con el fin de utilizarlas como potenciales marcadores biológicos. El desarrollo combinado de ensayos funcionales y aproximaciones moleculares, proteómicas y genómicas ha llevado a elucidar aspectos relevantes de las micropartículas, siendo de interés su uso como dianas terapéuticas así como nuevos prometedores biomarcadores de la enfermedad aterotrombótica silente.

The gram-positive bacterium Streptococcus sanguinis is a member of human indigenous oral microbialflora and has long been recognized as a key player in the bacterial colonization of the mouth. S. sanguinis is also the most common viridians streptococcal species implicated in infective endocarditis. Although many studies have focused on two-component systems in closely related Streptococcus species such as S. mutans, S. pneumoniae and S. gordonii; the mechanism of the response regulator in S. sanguinis is still unknown. The ability of S. sanguinis to adapt and thrive in hostile environments suggests this bacterium is capable of sensing and responding to various environmental stimuli. The present study clearly demonstrates that a number of RR genes, SSA_0204, SSA_0217, SSA_1810, SSA_1794, and SSA_1842, in S. sanguinis are essential to the recognition and response to various environmental stresses. Results from this study also identified genes SSA_0260, SSA_0261, and SSA-0262, involved in acidic tolerance and suppressed by SSA_0204 response regulator.

Streptococcus sanguinis is a gram-positive facultative anaerobe that is indigenous to the oral cavity and a primary colonizer of the oral cavity. It serves as a tether for the attachment of several oral bacteria that colonize the tooth surface, form dental plaque, and cause periodontal disease. Previous experiments with streptococcal strains have suggested that cellular chain morphology of streptococci may influence the competitiveness, susceptibility to phagocytosis, acidurance, and aggregation of the bacterium. The purpose of this study was to systematically determine gene functions that contribute to cellular chain length morphology in the SK36 strain of S. sanguinis. Gene functions for 2048 mutants were elucidated along with Clusters of Orthologous Groups (COG) functions that may be related to or regulate chain formation and morphology. The COG functions with high ratios of genes involved with chain length morphology per number of total non-essential mutant COG functions were in the following order: Cell division and Chromosome Separation, Defense Mechanisms, and Signal Transduction Mechanisms, and Cell Motility and Secretion. Examination of gene annotations of the 326 mutants involved with chain morphology suggests that cellular chain length is dependent on cell wall division and septation, peptidoglycan synthesis, and cell wall mobility. Some of the genes that contribute to chain length properties may be co-regulated which may suggest that chain length phenotypes are a transcriptionally regulated property that further studies may confirm.

The bacterium Streptococcus sanguinis is a primary member of the human oral microflora and also has been recognized as a key player in the bacterial colonization of the mouth. It is considered the most common viridians streptococcal species implicated in infective endocarditis. In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. Sequencing of the SK36 genome provided the opportunity to study ABC transporter mutants and their relationship with acidity of the oral environment. Despite numerous studies that have focused on carbohydrate uptake systems in closely related streptococcal species such as S. mutans, S. pneumonia and S. pyogenes, the mechanism of the response of these ABC transporters to acidic conditions in S. sanguinis is still unknown. The capability of S. sanguinis to adapt in these harsh environments suggests this bacterium is capable of responding to various environmental stimuli. The purpose of this study was to examine ABC mutants to identify functions that contribute to acid tolerance in S. sanguinis. This study demonstrates that two acid-sensitive mutant genes, SSA_1507 and SSA_1508, identify genes involved in acid tolerance. The two mutants grew on different sugars and none of them showed a defect in sugar utilization at acid pH. We couldn’t recognize any significant differences in sugar uptake for the two acid sensitive mutants or in mutants of their neighboring genes. Thus, the observed acid sensitivity is not due to a failure to take up any of the common sugars tested. The cytoplasmic pH of S. sanguinis was studied with the fluorescent pH indicator (BCECF) and SK36 was observed to have a wider pH range than either of the two acid-sensitive mutants SSA_1507 or SSA_1508. In these two mutants, intracellular pH was not as well maintained. At all pH values tested, the mutants displayed a lower intracellular pH than the wild type. These observations indicate that the cell membrane of these two mutants is unable to protect the interior components from adverse effects of higher pH values and lower pH values, and prove that these two mutant genes SSA_1507 and SSA_1508 are unable to grow in lower pH values. These results support a role for these ABC transporters in proton pump or export and indicate that the mutants are less able to pump out protons.

Streptococcus sanguinis is an important component of the dental plaque biofilm and is believed to play a beneficial role in the oral cavity. S. sanguinis is also a leading cause of infective endocarditis (IE), a potentially lethal infection of the cardiac valves. S. sanguinis possesses genetic competence, the ability to acquire exogenous DNA into its genome. In the well characterized system of S. pneumoniae, genetic competence requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternate sigma factor, ComX. Previous studies in other streptococcal species have suggested functions for the com regulon apart from DNA uptake. Here we characterized functions of the S. sanguinis com regulon genes in genetic competence, IE virulence, and biofilm formation. Our findings indicated that the early regulatory genes and those under the control of ComX in S. sanguinis play similar roles in genetic competence as their orthologs in other competent streptococci; however the sequence and mechanism of processing of the quorum sensing signal, competence-stimulating peptide, CSP, were determined to be unique. Using a rabbit endocarditis model, we determined that the comCDE and comX genes were not required for virulence, bacteremia, or pathology under a variety of infection conditions. In contrast, examination of biofilms by microscopy and crystal violet staining indicated that S. sanguinis CSP enhanced biofilm formation in a comDE-dependent manner. Deletion of the early com gene SSA_0195 eliminated this effect, while expression of the gene from an inducible promoter increased biofilm formation in the absence of CSP. Deletion of the comX gene resulted in biofilms with increased staining, cell death, and profoundly altered structure. Treatment with DNase I reduced biofilm formation in a com-independent manner. Taken together, these results suggest that expression of SSA_0195 is both necessary and sufficient for CSP-dependent biofilm enhancement, and that the late gene activator, ComX, is required to maintain normal biofilm architecture. Our findings suggest the com regulon of S. sanguinis may be an important determinant of competitiveness in the mouth, where native CSP production may occur at levels sufficient to influence biofilm formation.

The ability of bacteria to assimilate free DNA from the environment is known as competence. Though many studies have focused on competence regulation in Streptococcus pneumoniae and Streptococcus gordonii, Streptococcus sanguinis has yet to be examined. Physiological characterization of competence in S. sanguinis strain SK36 and its comC mutant, JFP41, led to the genome-wide transcriptional analysis of cells induced to competence via addition of competence-stimulating peptide (CSP). A total of 128 genes were induced at least 2-fold, 74 of which were classified as either “early” or “late” based on their induction patterns. Expression patterns were verified using qRT-PCR. This study identified genes not up-regulated in S. pneumoniae or S. gordonii and lays the foundation for bioinformatic studies to identify conserved binding sites upstream from CSP-regulated genes. These results also shed light on the possible existence and identity of expected CSP exporters in S. sanguinis, which have so far eluded discovery.

Dins de les mesures en la ona espectral intrarenal, l’índex de resistivitat (IRI) és el més conegut i estudiat com relacionat amb la valoració de la disfunció renal i el seu pronòstic. Així mateix s’ha relacionat amb marcadors de lesió d’òrgan diana i risc Càrdio-Vascular (RCV). Es realitza un estudi observacional de tall transversal per relacionar IRI amb variables de dany renal, RCV i rigidesa arterial. S’han estudiat 229 individus. L'IRI s’ha trobat significativament augmentat en pacients DM2 i amb insuficiència renal.S’ha correlacionat positivament amb l'edat i paràmetres de disfunció renal. A més s’han trobat correlacions positives i significatives amb tots els paràmetres de rigidesa arterial (VOP, AASI i pressió de pols de 24h), amb la disfunció endotelial, així com amb la càrrega ateromatosa (GIM i ITB). Els factors independents per a un IRI augmentat han estat l'edat, l'FG, la tensió arterial diastòlica de 24h, l'HbA1c i la rigidesa arterial. En resum, l’IRI es troba més elevat en diabètics i insuficients renals, i s’associa amb factors de risc càrdio-vasculars, amb rigidesa arterial i amb dany renal. / Among the intrarenal spectral wave measurements, the resistivity index (RRI) is the most known and studied as related to the assessment of renal function and its prognosis. Also, it has been associated with markers of target organ damage and cardiovascular risk. Cross-sectional and observational study in order to relate RRI with kidney damage, cardiovascular risk and arterial stiffness. We studied 229 individuals. RRI has been found to be significantly increased in DM 2 and in kidney disease. Intrarenal resistivity has been positively correlated with age, parameters of renal dysfunction. Also, positive and significant correlations have been found with all parameters of arterial stiffness (VOP, AASI and pulse pressure was 24), with endothelial dysfunction as well as atherosclerotic burden. Independent factors for increased RII were age, GFR, 24 h diastolic blood pressure, the HbA1C and arterial stiffness. In summary, the RRI is elevated in patients with diabetes 2 and renal impairment, and is associated with cardiovascular risk factors, arterial stiffness and kidney damage.